Specific chemical, biochemical and immunochemical assays have found widespread application in the fields of biomedical research and clinical diagnostics where they are used to determine the presence or amount of a variety of substances (analytes) commonly encountered in biological fluids. Such substances may include proteins, drugs, hormones, metabolites, nutrients, vitamins, microorganisms, etc. In addition, such specific assays may find utility in other fields, such as food processing and environmental quality control, for example, the detection of microorganisms and their toxins, or for detecting organic wastes. Specificity is very important because of the plethora of substances which may be present in samples.
Such specific assays are commonly divided into "chemistries" (chemical and biochemical assays) and binding assays (e.g. immunoassays). Such assays are further classified as "homogeneous" or "heterogeneous." In a homogeneous assay, a single reaction solution containing the sample is incubated to develop a detectable signal. For homogeneous binding assays, in which the amount of a bound label must be detected, the signal emitted by the bound labeled component is different from the signal emitted by the unbound labeled component. Hence, the two can be distinguished without the need for a physical separation step. The classical homogeneous specific binding assay is the enzyme-multiplied immunoassay technique (EMIT), described in U.S. Pat. No. 3,817,837, issued to Rubenstein.
Homogeneous assays are typically rapid and easy to perform, usually requiring ten minutes or less to complete. They can be carried out either manually or with automated instruments. Various methods for homogeneous assays are described in Methods in Clinical Chemistry, A. M. Pesce and L. A. Kaplan editors, C. V. Mosby Co., 1987.
Homogeneous immunoassays can be more complex to perform than other assays, in that they typically require sequential additions and mixing of reagents with careful timing. Automation is preferable and has been achieved with various large clinical analyzers (e.g. DuPont aca.TM., Roche Cobas Bio.TM.).
In spite of their simplicity, homogeneous assays have several disadvantages: they are prone to interferences, and are generally limited in sensitivity to detection of approximately one nanomolar analyte. Binding assays are the most specific homogeneous assays, but they are still subject to interferences, they are typically only compatible with low molecular weight analytes, and they require extended incubations (20-40 minutes) to detect nanomolar levels of analyte.
Heterogeneous assays are usually binding assays. In many such assays, both large and small molecules can be detected. At least one labeled ligand is present and a second phase (typically solid) is employed to separate bound from unbound label. Since the signal emitted by the bound and unbound labeled ligands is identical, the two must be physically separated in order to distinguish between them.
The classical heterogeneous specific binding assay is the competitive radioimmunoassay (RIA), described by Yalow (Science 200: 1245, 1978). Other heterogeneous binding assays are the radioreceptor assay, described by Cuatrecasas (Ann. Rev. Biochem., 43: 109-214, 1974), and the sandwich radioimmunoassay, described by Wide (pp. 199-206 of Radioimmunoassay Methods, edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970). Heterogeneous binding assays can be significantly more sensitive and reliable than homogeneous assays: interferences are usually eliminated, signal-to-noise ratios are improved because unbound label is eliminated, and excess binding reagents can sometimes be used to speed binding reactions involving very dilute analyte.
In a typical heterogeneous ("double antibody") competitive RIA, a known amount of radiolabeled ligand and ligand present in the sample compete for a limited amount of antibody. Sufficient time is allowed for specific binding to occur, after which the antibody and bound ligand are precipitated by addition of anti-immunoglobulin, washed to remove unbound label by repeated centrifugation, and the amount of labeled ligand present in the bound phase is determined. Heterogeneous competitive binding assays work equally well for low and high molecular weight substances.
A sandwich assay can be used to achieve greater sensitivity for analytes such as antigen in an immunoassay. In a sandwich assay, excess ligands are used to force binding at concentrations below the dissociation constant of the binding pair. Such assays usually employ a solid phase consisting of a plastic head to which an antibody is permanently attached. In the typical sandwich immunoassay, two antibody types are required, each of which can bind simultaneously to the antigen. The antibody which is not bound to the solid phase is labelled. As with competitive RIAs, one or more discrete washing steps to separate bound and unbound label are required, and sequential addition of reagents is typical. Sandwich assays are typically used for high molecular weight substances.
Because in heterogeneous assays the solid phase must be isolated and washed, and because sequential reagent additions are frequently required, they tend to be time consuming and labor-intensive. However, they offer desirable results because they can be used for low and high molecular weight compounds, are less prone to interferences than homogeneous assays, and can be sensitive to subpicomolar antigen concentrations. Automation of heterogeneous immunoassays has been accomplished with limited commercial success (ARIA II by Becton Dickinson, CentRIA by Union Carbide). Hunter describes such an automated device in U.S. Pat. No. 4,125,375 (issued Nov. 14, 1978). However, these devices have required either sophisticated and expensive instrumentation to carefully control liquid flow and to monitor bound and unbound fractions, or it has resulted in the detection only of the unbound label flowing through a rapidly hydrated antibody solid phase.
Several attempts have been made to eliminate the inconvenience of washing steps in heterogeneous minding assays. For example, Clover et al., GB 1,411,382, describe a method for measuring the amount of unbound radiolabel, after partial separation from bound label, by shielding the bound (lower) phase. However, it is well known in the art that the sensitivity and precision of specific binding assays is severely limited if changes in the unbound rather than the bound labeled component are measured. Furthermore, methods which lack a washing step have the disadvantage of detecting both tight-binding (specific) and weak-binding (nonspecific) label, resulting in very high nonspecific signal. Charlton et al., U.S. Pat. No. 4,106,907, issued Aug. 15, 1978, disclose another container for radioactive counting which consists of a tapered reaction tube having a radiation shield extending up from the bottom of the tube to a uniform height, such that only radiation from the supernatant (the unbound labeled fraction) can be detected. This method is subject to the same limitations as Glover et al., supra.
Chantot et al., Analyt. Biochem. 84:256, 1978, describe a radioreceptor assay method for measuring the binding of radiolabeled ligands to membrane receptors. The technique involves counting the total amount of radiolabel present, centrifuging the sample, and recounting with an externally mounted copper screen which serves to absorb radiation from a defined volume of the supernatant. The screen itself consists of a copper sleeve mounted on the outside of a custom-made test tube having a small knob precisely positioned above the base to support the screen. This method suffers from the disadvantage of requiring double detection, and suffers as well from high nonspecific binding as described above for the Glover and Charlton methods. Furthermore, the tube as disclosed is vulnerable to jamming and breaking in standard gamma counters. As with the above-described "screening" methods, the large diameter of the screen allows significant scattered radiation from within the screened volume to impinge on the detector, resulting in inaccurate measurements of the unscreened label. Also, because bound label is directly adjacent to and in contact with unbound label, normal and unavoidable variability in the position of the screen or in the volumes of the unbound and bound phases can cause significant variability in signal.
Bennett et al., (J. Biol. Chem. 252: 2753, 1977) describe a radioreceptor assay in which, after mixing and incubating reagents, the asssay mixture is transferred to a centrifuge tube to wash the solid phase containing bound label. They employed prolonged (30 minutes) high speed centrifugation to force the solid phase into a solution of 20% sucrose, followed immediately by freezing the assay tube in liquid nitrogen and excising the tip of the tube containing the solid phase and bound label. This method provides more effective separation of bound and unbound label than those described above, but has several significant disadvantages. The assay mixture cannot be incubated in situ on top of the sucrose solution, thus requiring separate incubation and separation vessels, because reactants would diffuse into the solution. Care must be used in loading the assay mixtures onto these sucrose solutions because mixing will cause dilution of the assay mixture, thus changing the equilibrium for assay reactants. The separation is relatively lengthy, and assay tubes must be frozen immediately after centrifugation because the bound label can dissociate from the solid phase and diffuse away from the tip of the separation tube. Finally, excising the tip of separation tubes is inconvenient, time-consuming, difficult to perform reproducibly, exposes the user to the risk of liquid nitrogen burns and radioactive contamination from fragments of frozen tubes and their contents, and would be very difficult to automate.
In U.S. Pat. No. 4,125,375 (issued Nov 14, 1978), Hunter describes a method and automated instrumentation for performing heterogeneous immunoassays by carefully injecting a sucrose solution underneath a previously equilibrated immunoassay mixture containing particles of higher density than the sucrose solution. The particles are allowed to settle into the injected subphase, thereby separating the particles from the unbound label. This method potentially eliminates some of the disadvantages inherent in the Bennett et al. method, but suffers from several significant shortcomings. These shortcomings include that: (1) it requires separate preequilibration of the assay mixture prior to separation of bound and free label, plus removal of liquid waste, and thus cannot be self-contained, (2) the method is not readily adaptable to the most rapid (centrifugal) separations, (3) it suffers from potential dilution and diffusion artifacts as in the Bennett et al. method, (4) it is not suitable for convenient and reproducible manual assays, and (5) any automated instrument would require pumps, tubing, plus reservoirs for reagents, wash solution, and liquid waste.
Linsley et al., Proc. Natl. Acad. Sci. (USA) 82: 356,1985, describe a radioimmunoassay for type I transforming growth factor in tissue cultures. The solid phase is S. aureus, and the bound label is separated from the unbound label by rapid sedimentation into a solution of 10% sucrose, followed by freezing in liquid nitrogen and excision of the tip of the centrifuge tube to determine the sedimented bound label. This method is essentially an immunoassay embodiment of the radio-receptor assay described by Bennett et al., with the inherent disadvantages of the former method.
Although each of the binding assay methods described above have brought minor improvements to the state of the art, there remains a need in the art for a method of specific binding assay which combines the ease and rapidity of homogeneous techniques with the enhanced sensitivity typical of heterogeneous techniques, for both isotopic and nonisotopic applications, without the undesirable variability, delay, labor, and dissociation which occur during the wash steps. Further, the method should allow rapid separations, should be convenient for manual use with standard detection instruments, and should be readily adaptable to semi-automated or fully automated instrumentation. Ideally the method should be self-contained, have minimal plumbing and moving parts, and be compatible with fully predispensed reagents.
Furthermore, there is a growing need for compact and inexpensive instrumentation for use in decentralized test sites (e.g. physicians' offices). Preferable features for testing systems adapted to decentralized, low volume sites include: (1) the use of unprocessed samples (e.g. whole blood); (2) elimination of instrument calibration and maintenance; (3) unit-dispensing with no multi-use reservoirs for reagents or waste; (4) containment of waste within a sealed reaction vessel for protection from infectious samples; (5) precision and accuracy attainable by unskilled users which is equivalent to that attainable on larger, automated clinical lab analyzers by skilled operators. Advances and available products designed to meet the needs of decentralized test sites are reviewed by J. A. Jackson and M. E. Conrad in American Clinical Products Review (Aug., 1987). However, no product is disclosed which solves the problems discussed above or provides these needed features. The present invention fulfills the needs described above for both homogeneous and heterogeneous assays and further provides other related advantages.